Intravenous tissue plasminogen activator (tPA) is an effective treatment for acute ischemic stroke, but nearly two decades after FDA approval, its utilization remains low, and up to 80% of patients who receive tPA show no beneficial response. Currently, neither the mechanisms underlying tPA's effects in human blood nor the reasons for most patients' failure to respond are well understood. In order to develop clinical methods of monitoring tPA's activity in blood in real time, to guide treatment for individual patients, we aim to better understand tPA's clinical effects, especially with respect to its pleiotropic signaling within the blood. We hypothesize that tPA efficacy has a measurable and quantifiable signature in the circulation, and that perturbation of a delicate balance in coagulation and thrombolysis pathways is related to poor efficacy. Comparing tPA responders vs. non-responders, we employ a combined approach including conventional as well as innovative mass spectrometry (MS) techniques. As points of entry, we focus on two aspects that our preliminary data suggest are pivotal: the ADAMTS13/vWF pathway which plays a role in clot formation and lysis and is affected by tPA, and glycated albumin, which may sequester tPA leading to poorer outcomes for diabetic patients. Aim 1: Characterize tPA response by exploring the ADAMTS13/vWF axis/pathway, measuring levels of ADAMTS13 and vWF, and assessing ADAMTS13 activity by means of ADAMTS13-specific vWF substrate fragments, in responders vs. non-responders. We hypothesize that tPA responders will have elevated ADAMTS13 levels and activity, and decreased vWF. Aim 2: Investigate non-response in diabetic stroke patients by exploring the role of glycated albumin, high levels of which are characteristic of diabetes. We hypothesize that glycated albumin is higher in nonresponders, and that glycated albumin sequesters tPA, thereby diminishing tPA's efficacy in diabetic patients. We will use innovative proteomic techniques to examine the molecules stuck to albumin in individual patients' blood, and we hypothesize that glycated albumin sequesters more tPA than non-glycated albumin. With three collaborating US centers (MGH, BWH, UMass Medical School), and a fourth separate confirmation cohort (Vall d'Hebron Hospital, Barcelona) - we are able to enroll patients at a pace sufficient for our most conservative power calculation based on preliminary data. Our encouraging pilot data, IRB-approved published SOP and detailed analysis plan for longitudinal measures of outcome, adjusting for multiple co-morbidities, demonstrate the feasibility of recruitment and analysis, and directly support our hypotheses and approach.